Many diseases and conditions benefit from therapeutic promotion of vasodilation. Among these, cardiovascular disease (CVD) is currently the leading cause of death and is predicted to be the number one cause of disability worldwide by 2030 (World Health Organization 2014). In the United States, approximately 1 of every 4 deaths from 1999-2015 was due to CVD (Centers for Disease Control and Prevention, 2016). Ischemic heart disease and stroke were the leading primary causes of premature death. The overall economic impact of CVD was estimated to be $200 billion annually in the U.S., and is expected to increase in future decades. More effective diagnostic tools and therapies are necessary to limit the growing burden of CVD in the U.S. and worldwide, particularly the diseases which manifest in unwanted clotting within the arteries of the heart or brain.
A major contributor to acute cardiovascular events and sudden deaths is the development of atherosclerotic plaques, a progressive thickening of the arterial wall due to the accumulation of cholesterol. Rupture of atherosclerotic plaques can form thrombi that occlude blood flow, potentially leading to a life-threatening event. Thrombi occurring in the coronary artery can lead to a heart attack, and in cerebral arteries can lead to ischemic stroke.
The early detection and treatment of CVD is vital to assess the risk of vulnerable plaques leading to an acute cardiovascular event. However, screening for vulnerable atherosclerotic plaque using current imaging modalities poses specific challenges. Direct visualization using noninvasive imaging methods, e.g. carotid ultrasound, cardiovascular computed tomography, magnetic resonance imaging, and positron emission tomography, are preferable for early diagnosis of vulnerable atherosclerotic plaque in high-risk patients. For example, carotid ultrasound with measurement of the intima-media thickness within the artery wall offers a way to diagnose the extent of subclinical atherosclerotic vascular disease, assess risk, and may offer a means to identify disease progression and monitor the effectiveness of preventive therapies. The use of microbubble based ultrasound contrast agents as a complementary tool to enhance vascular ultrasound imaging, known as contrast-enhanced ultrasound imaging, is emerging as an important method in facilitating the detection and characterization of atherosclerotic disease.
The use of microbubbles as ultrasound contrast agents (UCAs) in vascular imaging is well established. Most commercially available UCAs consist of gas-filled microbubbles which have mean diameters between 1-5 μm and are encapsulated with a protein, polymer, or lipid shell. Albunex® (GE Healthcare) was the first UCA approved by the U.S. Food and Drug Administration and consisted of an air-filled microbubble encapsulated by an albumin shell. Second generation UCAs such as Optison® (GE Healthcare), Definity® (Lantheus Medical Imaging) and Lumason® (Bracco Diagnostics, Inc.) contain high-molecular-weight gases (e.g. C3F8 and SF6 respectively), which have lower solubility in blood and thus increase the lifetime of the microbubbles in circulation. The low density and high compressibility of the gas core in UCAs enables efficient ultrasound scattering. Thus, the injected agents are acoustically responsive, or echogenic, and function as intravascular tracers which can be visualized using ultrasound.
In addition to traditional contrast-enhanced ultrasound imaging, there has been recent interest in advancing the applications of UCAs for molecular imaging of atherosclerosis. Molecular imaging techniques with targeted UCAs are being used increasingly for noninvasive diagnosis of inflammation, thrombus, and neovascularization. Targeted microbubble agents are also being developed for controlled drug-delivery applications and have been vigorously promoted for therapeutic applications in the treatment of CVD. Targeted UCAs are functionalized by engineering the gas-encapsulating shell to contain molecules that adhere to cells, which express disease-specific markers (e.g., aminoacids) on the membrane. Phospholipid-shelled UCAs are of particular interest for this purpose, because they can be targeted to molecular components of disease by attaching specific ligands to the surface.
Phospholipid-shelled UCAs represent one type of UCA that is currently available for clinical use. The lipid molecules employed in the formulations are typically amphiphilic molecules which spontaneously form micelle structures that can encapsulate a gas microbubble in an aqueous environment. The lipids are surface-active molecules (surfactants) that orient their hydrophilic polar groups outside towards the surrounding aqueous medium and their hydrophobic tails inside away from the water, stabilizing the microbubble and largely preventing the gas from escaping the encapsulation. Lipid-based ultrasound contrast agents such as Definity® and Lumason® (which was recently approved for clinical use in the U.S. but has been marketed as SonoVue® in Europe and Asia since 2001) are commercially available for diagnostic applications. MicroMarker® (VisualSonics, Toronto, Canada; Bracco Research SA, Geneva, Switzerland) and Targestar® (Targeson Inc., San Diego, Calif., USA) are examples of targeted phospholipid-shelled UCAs currently available for pre-clinical investigational use.
A more recent formulation in the broad category of phospholipid-shelled UCAs, known as echogenic liposomes (ELIP), has been developed which encapsulates both a gas and an aqueous phase (Alkan-Onyuksel et al. 1996; Huang et al. 2001). Standard liposomes are characterized by a phospholipid bilayer shell, which encapsulates an aqueous compartment. ELIP are said to be echogenic because they contain a gas microbubble that is highly reflective to ultrasound waves at low intensities. The exact location of the entrapped gas pockets in ELIP has not been fully ascertained, and may be due to gas pockets stabilized by lipid monolayers within the liposome, or within the lipid bilayer shell.
Targetable drug-delivery systems represent a fast developing area of nanotechnology and are expected to have a dramatic impact on medicine in the future. Many nano-scale drug carriers, such as liposomes, micelles, and polymer nanocapsules, have been developed or are under development for encapsulation and delivery of therapeutic drugs. Liposomes are a convenient, biologically compatible vehicle for administration of poorly soluble drugs, and are among the first generation of nano-scale drug delivery systems to be approved for clinical use and known as nanomedicines (Moghimi et al. 2005).
Gregoriadis and Ryman (1971) were the first to report on the use of liposomes as drug carriers for directed delivery. The authors hypothesized that encapsulation of enzymes within the aqueous inner compartment of liposomes would aid in directing the payload to a particular tissue and alleviate some of the problems associated with immunological response to the proteins in circulation. They found that liposomes remain largely intact during circulation and are cleared by lysosomes in the liver (and to a lesser extent in the spleen). Since then, liposome based drug-delivery systems have been developed using chemotherapeutic agents for cancer therapy, thromolytic agents, and genes, in addition to enzymes.
Most of the currently approved liposome formulations represent a basic form of nanomedicine involving a passive targeting and drug release process known as the enhanced permeability and retention (EPR) effect. This approach relies on extravasation and accumulation of the liposome-encapsulated drug at the target site, and is particularly suited for cancer therapy applications due to the enhanced vascular permeability of tumors compared with normal tissue. Because tumors are highly vascularized and often lack effective lymphatic drainage, liposomes tend to accumulate in tumors much more than they do in normal tissues, resulting in increased drug uptake in these regions. Although EPR is a rudimentary passive targeting method, it is a key reason liposomes are currently the most widely used drug nanocarrier in cancer therapy. To realize the drug delivery potential of liposomes for other applications fully, however, it is important to develop agents with an active triggering mechanism that allows the drug to be delivered in a more controlled fashion. Echogenic microbubbles, by virtue of their ability to encapsulate gas as well as therapeutic drugs, offer such a possibility.
Recently, ultrasound has been investigated as a method to trigger enhanced drug delivery within the human vasculature. The potential of ultrasound to control drug delivery spatially and temporally in a non-invasive manner is broadly appealing. Ultrasound-mediated drug delivery (UMDD) has been demonstrated in a number of tissue beds, for example the blood-brain barrier, cardiac tissue, prostate, and large arteries.
Acoustic cavitation is one physical mechanism that is hypothesized to influence UMDD. Cavitation as used herein refers to linear or nonlinear bubble activity that can occur near vessel walls within the vasculature upon ultrasound exposure, which can exert mechanical stress on nearby cells and junctions. Mechanical stress can disturb the barriers to drug delivery such as endothelial tight junctions or phospholipid membranes, via transient permeabilization. In vivo, cavitation can be nucleated at moderate acoustic pressure amplitudes (<0.5 MPa) by ultrasound contrast agents (UCAs).
Nitric Oxide (NO) is a gas molecule that dynamically modulates the physiological functions of the cardiovascular system, which include relaxation of vascular smooth muscle, inhibition of platelet aggregation, and regulation of immune responses. Because a reduced NO level has been implicated in the onset and progression of various disease states, NO is expected to provide therapeutic benefits in the treatment of cardiovascular diseases, such as essential hypertension, stroke, coronary artery disease, atherosclerosis, platelet aggregation after percutaneous transluminal coronary angioplasty, and ischemia/reperfusion injury. To date, pharmacologically active compounds that can release NO within the body, such as organic nitrates and sodium nitroprusside, have been used as therapeutic agents, but their efficacy is significantly limited by their rapid NO release, poor distribution to the target site, toxicity, and induction of tolerance. Attenuation of nitric oxide production in the etiology of atherosclerosis progression and diabetic vascular disease further highlights the need for novel therapeutic nitric oxide modulation and delivery strategies. Effective delivery of bioactive NO to target cardiovascular tissue remains a compelling need in the art.
Xenon is a gas molecule that induces robust cardioprotection and neuroprotection through a variety of mechanisms. Through its influence on Ca2+, K+, KATP/HIF, and NMDA antagonism, xenon is neuroprotective when administered before, during and after ischemic insults. Xenon has particular promise as a bioactive agent for the treatment of cerebrovascular diseases and conditions. However, effective delivery of bioactive xenon to target cardiovascular or cerebrovascular tissue remains a need in the art.
Hydrogen sulfide is an endogenously produced gasotransmitter involved in the regulation of nervous system, cardiovascular functions, inflammatory response, gastrointestinal system, and renal function. Hydrogen sulfide gas has therapeutic potential for diseases such as arterial and pulmonary hypertension, atherosclerosis, ischemia-reperfusion injury, heart failure, peptic ulcer disease, acute and chronic inflammatory diseases, Parkinson's and Alzheimer's disease, and erectile dysfunction. Effective delivery of bioactive hydrogen sulfide to target diseased sections of vasculature remains a need in the art.
Hence, the need exists for improved compositions and methods for targeted ultrasound-mediated delivery of therapeutic bioactive gases.